Cryptopain compositions for prophylaxis, treatment, diagnosis and detection of cryptosporidium species

ABSTRACT

Vaccines, antibodies, proteins, DNAs and RNAs for diagnosis, prophylaxis, treatment and detection of  Cryptosporidium  species or  Cryptosporidium  species infections.  Cryptosporidium  species antigen and DNAs and RNA encoding the  Cryptosporidium  antigen and fragments thereof and recombinant proteins or fusion proteins produced thereby. Methods for diagnosis, prophylaxis, treatment and detection of  Cryptosporidium  species infections.

[0001] This application is a based on the provisional application Ser.No. 60/014233 filed on Mar. 27, 1996.

[0002] This invention was developed partially with U.S. Governmentsupport under National Institutes of Health Grant No U01-AI35123. TheU.S. Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] This invention concerns vaccines, antibodies, proteins, DNAs andRNAs for diagnosis, prophylaxis and treatment of Cryptosporidium speciesinfections and for detection of Cryptosporidium species. In particular,this invention concerns Cryptosporidium species antigen comprised of aprotein, as well as polyclonal and monoclonal antibodies directedagainst the antigen, DNAs and RNA encoding the Cryptosporidium speciesantigen and fragments and analogs thereof, and methods for production ofrecombinant or fusion proteins. This invention also concerns methods fordiagnosis, prophylaxis, treatment of Cryptosporidium infections anddetection of Cryptosporidium species.

BACKGROUND AND RELATED DISCLOSURES

[0005] The genus Cryptosporidium consists of Apicomplexan parasites thatinvade and develop within epithelial cells of the gastrointestinal,hepatobiliary and respiratory tracts-of a wide variety of vertebratesincluding reptiles, birds and mammals. Cryptosporidium was recognized asa cause of animal disease for several decades before the first cases ofhuman cryptosporidiosis were reported in 1976. However, it was not until1982 that the magnitude of disease caused by this parasite in both AIDSpatients and immunocompetent hosts began to be appreciated.Subsequently, Crytosporidium has been found to be one of the most commoncauses of human diarrhea worldwide, and to be an increasingly recognizedcause of diarrhea in children, animal care workers, and travelers.(Cryptosporidium and Cryptosporidiosis in Humans, Ed. Fayer, R., CRCPress, Boca Raton (1997)).

[0006] Large waterborne outbreaks of cryptosporidiosis caused bycontaminated municipal water supplies in the US or in the UK have beennoted in the last ten years (N. Engl. J. Med., 320:1372 (1989), and33:161 (1994)). The most recent outbreak in Milwaukee in April 1993involved 400,000 persons and led to the subsequent deaths of more than100 immunocompromised persons. Like a number of other waterborneoutbreaks, the Milwaukee outbreak appears to have been due tocontamination from farm or abattoir run-off and specifically tocryptosporidiosis among cows/calves. Nosocomial transmission inhospitals from patients to staff, patient to patient, and contaminatedice to patients and staff have also been well documented (J. Infect.Dis., 158:647 (1985)).

[0007] Waterborne and nosocomial spread uncovered a number of biologicalcharacteristics of oocysts. First, the infectious dose of a parasite isvery low. The ID₅₀ for human volunteers with normal immune systems is132 oocysts N. Engl. J. Med., 332:855 (1995). Second, infected hosts,for example calves, excrete large numbers of oocysts, on the order of10¹⁰/day. Third, the oocysts are fully sporulated and ready to infectwhen excreted. Fourth, the oocysts are environmentally hardy. Theyremain infectious in cool, moist areas for 3-4 months. They are notkilled by chlorine levels achievable in drinking water. Fifth, theoocysts are quite small, 4-6 μm, and are thus difficult to filter.

[0008] The clinical importance of cryptosporidiosis has increasedmarkedly with the recognition of a life-threatening form of the diseasein patients with immunodeficiency disorders such as AIDS,hypogammaglobulinaemia, and chemotherapeutic immunosuppression. Theprevalence of cryptosporidiosis in AIDS patients in the US is estimatedto be 5-10% and in central Africa 40%. Immunodeficient patients may havefulminant cryptosporidial diarrhea that may persist until death, whereasthe diarrhea of immunocompetent patients is self-limited and rarelylasts more than 2-4 weeks. Cholera-like diarrhea is common inimmunocompromised patients with reported losses of up to 17 liters perday. Hepatobiliary disease may result in severe abdominal pain andnausea. Removal of immunosuppression in chemotherapy patients leads toresolution of the diarrhea. Some AIDS patients with cryptosporidiosiswill be able to eliminate the parasite by induction of anti-retroviraltherapy (Am. Intern. Med., 116:840 (1992)).

[0009] Among those who develop disease, a quarter have CD4 countsgreater than 209, suggesting that the disease may occur relatively earlyin the course of HIV disease (Am. J. Epidemiol., 144:807 (1996).Unfortunately, few details about the biology and molecular mediators ofthe disease process have been described and so far no effective therapyhas been discovered.

[0010] The infective forms of Crytosporidium, called sporozoites andmerozoites, appear to adhere to the host cell and release the contentsof anterior organelles (rhoptries, micronemes or dense granules) duringthe invasion process (Parasitol. Today, 8:28(1992)). Proteins involvedin these events have in many instances been found to be the target ofinvasion blocking immunity in vitro and neutralization in vivo (Infect.Immun., 56:2538(1988)).

[0011] Active and passive immunization studies using malaria andToxoplasma challenged or infected hosts have shown that certain secretedcomponents of the apical complex organelles are the target of protectiveantibodies. In some cases, as for example in the case of thecircumsporozoite and merozoite surface proteins of malaria, theseantigens are under development as vaccines.

[0012] While the actual interaction between Crytosporidium and thehost's immune system is poorly understood, it is known that disruptionof either the cellular or the humoral components can result inprotracted cryptosporidiosis (Parasitol. Today, 8:24 (1992)). However,specific antibodies alone appear to be enough to neutralize theorganism's infectivity. In vitro and in vivo observations indicate thatantibodies to Crytosporidium parvum inhibit invasion and intracellulardevelopment leading to protection in challenge experiments, oramelioration of infection in established disease (Infect. Immun.,59:1172 (1991)).

[0013] One source of such antibodies is hyperimmune bovine colostrum(HBC) collected from cows immunized with Crytosporidium oocysts. Calveschallenged with Crytosporidium oocysts are protected from thedevelopment of disease by the administration of HBC (Infect. Immun.,61:4079 (1993)). Some immunocompromised AIDS patients infected withCrytosporidium have also responded to HBC with a reduction in ordisappearance of the symptoms of the disease (Gastroenterology, 98:486(1990)). Immunoglobulin from HBC (HBC Ig) has been found to inhibit theability of the sporozoite to invade and/or develop intracellularly invitro and it has been used to immunoprecipitate at least 22 differentsurface radioiodinated proteins of Crytosporidium sporozoites. Westernblot analysis of proteins of whole oocysts which contain sporozoite,indicates that HBC predominantly recognizes two proteins of sizes 250 Kdand >900 Kd (Infect. Immun., 61:4079 (1993)).

[0014] The use of HBC for human use is problematic. HBC produced usingwhole oocysts is batch dependent and this may lead to the development ofpassive immune preparations which are not uniform in immunogenicity andpotency. This generates a problem when these immune preparation are tobe administered to human patients as such non-uniformity may result infailure of protection. In addition, it would be desirable to allowpreparation of large amounts of antigen expressed in heterologoussystems than to purify oocyst.

[0015] Thus, there is a continuous need for immunogenic agents which arereasonably reproducible and have uniform and controllable immunogenicityand potency which agents would be useful for the immunotherapy ofcryptosporidiosis in both uncompromised and immunocompromised subjects,such as AIDS patients, and would allow the prophylaxis and treatment ofcryptosporidiosis.

[0016] Additionally, there is a need to have available methods forreproducible expression of specific target for Crytosporidium antigen inlarge amounts, which antigen would provide a better immunogen. Thisapproach requires that a specific Crytosporidium antigen is cloned andidentified as a potential candidate through its ability to elicit anantibody response that is immunoprotective. Before antibodies producedin this manner are tested in or administered to humans or animals,testing in in vitro assay of their inhibitory effect on invasion orintracellular development of the Crytosporidium organism in culturedcells and in vivo studies would be desirable.

[0017] It is, therefore, a primary objective of this invention toprovide Crytosporidium cryptopain polyclonal or monoclonal antibodiesand vaccines to be used for prophylaxis, treatment, diagnosis anddetections of cryptosporidiosis and to express a portion of thecryptopain sequence/locus to provide target protein antigens allowingproduction of recombinant anti-Crytosporidium vaccines and passiveimmune products.

[0018] All patents, patent applications and publication cited herein arehereby incorporated by reference.

SUMMARY OF THE INVENTION

[0019] One aspect of this invention concerns vaccines, antigens,antibodies, proteins, DNAs and RNAs for prophylaxis, treatment anddetection or diagnosis of Crytosporidium species or Crytosporidiumspecies infections.

[0020] Another aspect of this invention concerns a Crytosporidiumantigen protein comprising pre, pro, and mature enzyme sequences andtheir fragments.

[0021] Still another aspect of this invention concerns polyclonal ormonoclonal antibodies directed against the Crytosporidium antigen.

[0022] Still yet another aspect of this invention concerns a DNA and RNAencoding the Crytosporidium antigen and fragments thereof and theantigen pre, pro, and mature regions.

[0023] Another aspect of this invention concerns a polyclonal ormonoclonal antibodies directed against invasive stages ofCryptosporidial species capable of preventing and ameliorating invasionof Crytosporidium infection.

[0024] Still another aspect of this invention concerns a natural,synthetic or recombinant vaccine useful for active immunization ofanimals and humans against Crytosporidium infection.

[0025] Still another aspect of this invention concerns a natural,synthetic or recombinant protein useful for preparation of passiveimmune products for treatment of established infection.

[0026] Another aspect of this invention concerns a natural, synthetic orrecombinant DNA vaccine capable of endogenous production of inhibitoryamount of anti-Crytosporidium parvum antibodies.

[0027] Another aspect of this invention concerns a natural, synthetic orrecombinant RNA vaccine capable of endogenous development of inhibitoryamount of anti-Crytosporidium parvum antibodies.

[0028] Still another aspect of the invention concerns a method for useof a pre pro enzyme portion of the cysteine proteinase molecule as acompetitive inhibitor of the action of the mature enzyme.

[0029] Still yet another aspect of the invention is the use of antigen,antibody, DNA or RNA to detect the presence of the cysteine proteinaseor antibodies to cysteine proteinase, or DNA or RNA encoding thecysteine proteinase, for diagnosis in a human or animal host ordetection in the environment.

[0030] Another aspect of this invention concerns the sequence of a 401amino acid protein comprising a cathepsin L-like cysteine proteinase ofMW 45 kDa present in sporozoites and merozoites, and its amino acid andsize variants including a deduced mature 226 amino acid protein of MW 25kDa.

[0031] Another aspect of this invention concerns the DNA sequence of1203 nucleotides encoding the 45 kDa protein, the cathepsin-likecysteine proteinase, cryptopain, its nucleotide and size variants andits upstream regulatory elements.

[0032] Another aspect of this invention concerns the RNA sequencedetermined by the DNA sequence of cryptopain and its nucleotide and sizevariants including polyadenylation sequence.

[0033] Still yet another aspect of this invention concerns a group ofcryptopain recombinant or expressed protein targets of polyclonalantibodies which inhibit Crytosporidium infection, invasion, oradhesion.

[0034] Another aspect of this invention concerns a method forprophylaxis and treatment of Crytosporidium or Crytosporidium infectionsusing vaccines, antibodies, proteins, DNAs and RNAs of the invention.

[0035] Still yet another aspect of this invention concerns a method ofprophylaxis, treatment, inhibition or retardation of a Crytosporidiuminfection comprising administering to a subject in need of suchtreatment an amount of an anti-Crytosporidium polyclonal or monoclonalantibodies prophylactically or therapeutically effective to provideimmunity against infection or treatment for disease.

[0036] Still yet another aspect of this invention concerns a method ofprophylaxis, treatment, retardation, or inhibition of Crytosporidiuminfection comprising administering to a subject in need of suchtreatment a vaccine comprising the polypeptide of this invention or itsDNA or RNA capable of endogenous stimulation of the production ofinhibitory amount of anti-Crytosporidium antibodies or protectivecellular immune responses.

[0037] Still yet another aspect of this invention concerns a method fordiagnosing Crytosporidium infection of a subject, comprising steps:

[0038] (a) contacting a body specimen, fluid or tissue obtained from thesubject with an anti-Crytosporidium monoclonal or polyclonal antibody;and

[0039] (b) detecting the formation of antibody-antigen complex whereinthe presence of the complex indicates the presence of a Crytosporidiumorganism in the subject.

[0040] Still yet another aspect of this invention concerns a method fordetecting anti-Crytosporidium antibody in a subject, said methodcomprising steps:

[0041] (a) contacting a body specimen, fluid or tissue obtained from thesubject with the cryptopain; and

[0042] (b) detecting a formation of antibody-antigen complex wherein thepresence of the complex indicates the presence of a Crytosporidiumantibody in the subject.

[0043] Still another aspect of this invention is a Crytosporidiumdiagnostic or detection kit comprising anti-Crytosporidium specificmonoclonal and polyclonal antibodies or antigen according to theinvention and a means for detection of an antibody-antigen complex.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044]FIG. 1 is a diagram showing the strategy model for developing aprobe for the Crytosporidium cysteine proteinase using consensusoligonucleotide primers for PCR amplification of genomic DNA. The modelis compared to previously published diagrams of the primary structure ofcysteine proteinases from other organisms.

[0045]FIG. 2 is the DNA sequence of cryptopain (SEQ ID NO: 1) comprisingsequences encoding segments for the pre and pro regions (SEQ ID NO: 2),mature enzyme coding region (SEQ ID NO: 3) and 3′ and 5′ flankingsequences.

[0046]FIG. 3 is the protein sequence of cryptopain (SEQ ID NO: 4)comprising segments for the pre and pro regions (SEQ ID NO: 5) and formature enzyme (SEQ ID NO: 6).

[0047]FIG. 4 is an amino acid alignment showing marked amino acidsimilarities of cryptopain to other cathepsin-like cysteine proteinases(SEQ ID NOs: 4, 7 and 8).

[0048]FIG. 5 shows a genomic Southern analysis of Crytosporidium DNAusing the cryptopain probe.

[0049]FIG. 6 shows a Kyte Doolittle hydropathy plot indicating anN-terminal hydrophobic sequence consistent with membrane targeting andsecretion of cryptopain.

[0050]FIG. 7 are oligonucleotide sequences used to generate DNAfragments of the cryptopain gene. FIG. 7Al is a degenerate primer basedon the conserved cysteine (sense) and FIG. 7A2 is a degenerate primerbased on conserved arginine (antisense) of the P. vinckei cysteineproteinase gene. These primers were used to amplify the 459 bp fragmentof cryptopain from C. parvum DNA. FIG. 7B shows primers used todirectionally clone the entire C. parvum gene comprising pre, pro andmature protein encoding regions, to be expressed as a thioredoxin fusionprotein. FIG. 7B1 is the sense and FIG. 7B2 is the antisenseoligonucleotide.

[0051]FIG. 8 is a diagram of pTrxFus showing the directional cloningstrategy.

[0052]FIG. 9 is a Western blot of cryptopain expressed as a thioredoxinfusion protein and detected by anti-thioredoxin antibody.

[0053]FIG. 10 are graphs showing percentage of invasion/ intracellulardevelopment of Crytosporidium parvum sporozoites in vitro in MDCK cellsin the presence of inhibitors of cysteine proteinases. FIG. 10A is acomparative graph of three cysteine proteinase inhibitors biotinylatedfluoromethylketone (BPAFMK) (FIG. 10B);trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane E64 (FIG. 10C);and proprietary compound K-111 (FIG. 10D). FIG. 10B-10D show standarddeviations.

DEFINITIONS

[0054] As used herein:

[0055] “Cryptopain” or “Crytosporidium antigen” means a protein which isa cathepsin L-like cysteine proteinase having a function in invasion andinfection of host cells by Crytosporidium. Cryptopain is represented bya protein containing 401 amino acids and is identified as SEQ ID NO: 4(FIG. 3) comprising a protein of MR 45 kDa. Homology to other cathepsinL-like cysteine proteinases seen in FIG. 4 indicates that the matureactive enzyme is cleaved after amino acid 175 one residue N-terminal toa conserved prolines and comprises a 25 kDa protein of 226 amino acids.Cryptopain also includes size and sequence variance proteins whichmaintain the same function.

[0056] The “structure” or “structural characteristics” of cryptopaindefines a protein, and DNA and RNA encoding the cryptopain protein andincludes all structural variations, mutations and fragments exhibitingthe same function.

[0057] The “functionality” or “functional characteristics” of cryptopainis defined by the action of the protein and structural variantsdescribed, such that infection and disease occurs.

[0058] “Inactive enzyme” means enzyme comprised either of mature enzymeregions and pro regions, or mature enzyme and pro and pre regionswherein the pro or pre pro regions are responsible for the mature enzymenonfunctionality or for the inhibition of its function.

[0059] “Active enzyme” or “mature enzyme” means functional enzyme and iscomprised of the mature region. Mature enzyme contains the catalyticactive sites of the cysteine proteinase and typically begins with oneresidue N-terminal to a conserved proline.

[0060] “Pro” or “pro region” means the contiguous amino acid sequencewhich renders the mature enzyme inactive by its structural associationwith it.

[0061] “Pre” or “pre region” means the terminal amino acid sequencewhich is contiguous with the pro region and may contain a signal fortrafficking movement of inactive enzyme in the cell.

[0062] “The gene” or “genes encoding cryptopain” means DNA encoding thecryptopain protein.

[0063] “Sporozoites or merozoites ” mean any life stage which may invadeor develop in the host cells and any variant or mutant of said lifestages.

[0064] “Antibodies” means proteins which structurally interact with thetarget antigen and are produced when the antigen is introduced into ananimal, such that they stimulate the immune system. The term alsoincludes antibodies produced in vitro, such as chimeras, or hybridomacell cultures, as well as hybridomas or chimeric constructs introducedinto a host to provide an in vivo antibody.

[0065] “Antibodies to cryptopain” means proteins which structurallyinteract with the target antigen cryptopain and inhibit invasion,infection or development of the sporozoites or merozoites in the hostcell.

[0066] “Monoclonal antibodies” means the monovalent antibodies producedby an B cell fused to an immortalized cell producing specific antibodyto cryptopain.

[0067] “Polyclonal antibodies” means antibodies directed at cryptopainwhich are not monovalent and are the products of multiple B cells incharacter.

[0068] “Crytosporidium antigen” means a protein with or withoutcarbohydrate attached thereto which defines a capacity of Crytosporidiumsporozoites and merozoites to infect and develop in host cells.

[0069] “Cryptopain DNA” means the sequence of 1203 polydeoxyribonucleotides identified in SEQ ID NO: 1 (FIG. 2) which encodes the aminoacid sequence of Crytosporidium antigen (SEQ ID NO: 4) and any variants,mutations and fragments thereof which correspond to or would detectgenes encoding the antigen and includes specific PCR oligonucleotideprimers for amplification of cryptopain sequences and fragments ofsequence used as genetic probes for detection of cryptopain sequence.Also included is DNA inserted into host cells for the purpose of in vivoexpression of target antigen in order to stimulate the host immunesystem.

[0070] “Cryptopain RNA” means the sequence of 1203 nucleotides whichencodes the protein sequence of cryptopain protein (SEQ ID NO: 4) (FIG.3) and any variants, mutations and fragments thereof includingpolyadenylation tail which correspond to or would detect genes encodingthe antigen. RNA probes and RNA inserted into host cells for the purposeof in vivo expression of target antigen in order to stimulate the hostimmune system are included.

[0071] “Vaccine” means protein, recombinant protein, DNA or RNA fromcryptopain which, upon introduction into a host, is able to provoke animmune response including but not limited to the production ofantibodies, cytokines and other cellular responses.

[0072] “Detection” means establishing or providing evidence for thepresence or prior presence of living or dead Crytosporidium by detectingcryptopain protein, Crytosporidium protein specific activity, DNA or RNAin the host, in a host tissue specimens, or in environmental samplesincluding water, soil, food, etc.

[0073] “Diagnosis” means establishment of the presence or prior presenceof Crytosporidium infection or disease by using the cryptopain protein,Crytosporidium protein specific activity, DNA or RNA as a component of adiagnostic assay according to the invention.

[0074] “Prevention or prophylaxis” means the immunization or vaccinationof the host with a vaccine of the invention such that Crytosporidiumdisease or infection does not occur.

[0075] “Treatment” means therapeutic use of any protein or antibody toinhibit Crytosporidium infection in a host.

[0076] “Host” or “subject” means human, or animal including birds andcattle.

[0077] “Regulatory elements” means nucleotide sequences which controlthe expression of genes they regulate, typically by interaction withother macromolecular species such as protein.

[0078] “Active immunity to infection” means ability of the organism toproduce specific responses such as production of cytokines, lymphokines,antibodies or other substances, or cellular capacity to inhibit orretard infection in response to a contact with antigen.

[0079] “Passive immunity to infection” means the transfer to a host ofthe specific antibodies or other substances or cells capable ofinhibiting or retarding infection.

[0080] “Crytosporidium species” means any organism belonging to thegenus Crytosporidium, such as, for example,

[0081]Crytosporidium parvum or Crytosporidium muris, but also includescurrently less well characterized other organisms such as, for example,Cyclospora and similar organisms, such as Eimeria. Crytosporidiumspecies comprise Apicomplexan parasites which primarily invade cells ofgastrointestinal tract and cause disease in a susceptible host.

[0082] “Recombinant vaccines” means DNA/RNA/protein segments propagatedor expressed in foreign system. This includes all vaccines other thanbiologically derived vaccines.

[0083] “Biologically derived vaccines” means vaccines made from aprotein or carbohydrate generated in the organism of origin.

DETAILED DESCRIPTION OF THE INVENTION

[0084] The current invention is based on findings that cryptopain, acathepsin L-like cysteine proteinase, localized at the Crytosporidiumsporozoites surface or within its cell, is involved in Crytosporidiuminfectivity and that such infectivity can be prevented by cryptopaininhibitors.

[0085] Cryptopain deduced amino acid sequence shows homology to othercathepsin L-like cysteine proteinases indicating that the mature activeenzyme is a 25 kDa protein of 225 amino acids. Cryptopain DNA has beenisolated, purified, sequenced and recombinantly produced. Cryptopainfusion protein in which the fusion partner is thioredoxin has also beenrecombinantly produced.

[0086] Due to its unique biological activity, cryptopain may beadvantageously used for prophylactic, therapeutic, diagnostic anddetection purposes.

[0087] This invention, therefore, relates to isolated native andrecombinantly produced cryptopain; cryptopain amino acid, DNA and RNAsequences; and to vaccines, antibodies, proteins and synthetic proteins,DNAs and RNAs useful for prophylaxis, treatment, diagnosis and detectionof infections caused by any Crytosporidium organism or any organismbelonging to Crytosporidium species.

[0088] More specifically, the invention concerns identification andcryptopain, of a Cryptosporidium antigen, comprised of a protein orpolypeptide, identification of DNA of the Crytosporidium antigen genewithin the locus, sequencing DNA encoding the Crytosporidium antigen,expressing portions of the locus encoding the Crytosporidium antigen andusing the expressed antigens for preparation of vaccines or forpreparation of polyclonal or monoclonal antibodies.

I. Cryptopain—Cryytosporidium Parvum Antigen

[0089] Cryptopain is cathepsin L-like cysteine proteinase. It isstructurally and functionally similar to other cysteine proteinases,represented, for example, by Carica papain and Plasmodium vinckeicysteine proteinase, and its activity is inhibited by group of cysteineproteinase specific inhibitors.

A. Cysteine Proteinases—Their Function, Structure and Inhibition

[0090] There are four major classes of proteinases for which thecatalytic mechanism has been defined. These proteinases are designatedcysteine, aspartic, metallo and serine proteinases. The major mammaliancysteine proteinases are the lysosomal cysteine proteinases, cathepsinsB, H and L proteinases and the cytoplasmic calpains. Mammalian cysteineproteinases B and L are also active at neutral pH, and are found outsidethe cell and may function in the degradation of extracellular proteins.The sequences of the protozoan cysteine proteinases identified to dateshow that they are more closely related to cathepsin L than to cathepsinB. Cysteine proteinases essentially contain amino acids cysteine,histidine and asparagine which are important for the action of theproteinases. The sulfonium ion of the cysteine provides the nucleophilicattack on the carbonyl group of the targeted peptide bond in order toeffect hydrolysis of the bond.

[0091] Calpains and cathepsins are generally distinguished from eachother by their cellular locations and by their inhibition profile. Forexample, cathepsins, but not calpains, are inhibited by the peptidyldiazomethane and peptidyl fluoromethylketone inhibitors Z-phe-ala-CHN₂(diazomethane) and Z-phe-ala-FMK (fluoromethylketone). Both lysosomalcysteine proteinases and calpains are inhibited by the class-specificinhibitor E64 and the more general inhibitor leupeptin.

[0092] Peptide inhibitors have been used to determine the peptide bondspecificity of proteinases. The specificity of the inhibitor isdetermined by the amino acid residues, for example, phe-ala residues,which bind in the pocket formed by the active sites of the enzyme.Peptide inhibitors only bind to active enzyme, i.e. enzyme which has aconformationally correct enzyme pocket. Peptide inhibitors are usefulfor detection of the presence of specific types of cysteine proteinasesin living systems as they may allow the localization or detection ofenzymatic activity in the absence of isolation and purification of theenzyme with the subsequent development of antibody probes. Sinceisolation of active enzyme by biochemical techniques requires largeamounts of material and the isolated enzyme is often not stable, use ofpeptide inhibitors instead is very advantageous.

[0093] Proteinase inhibitors are a new type of agent for treatment ofprotozoan infection. Cloning of genes for selected proteinases,expression of the proteinases, and molecular modeling of the proteinasesare techniques which have facilitated the development of cysteineproteinases inhibitors specific for a given enzyme, such as for example,falcipain of P. falciparum. In addition, the differences betweenmammalian and protozoan cysteine proteinases and between cysteineproteinases of specific protozoa allow development of detectiontechniques for the organism based on the acting of the enzyme, DNA, RNAand antibodies.

B. Cryptopain Gene Cloning, Sequencing and Genomic Southern Analysis

[0094] In order to provide consistently the same antigen for productionof antibodies or vaccines, and for recombinant production of fusionproteins and other agents useful for prophylactic therapeutic anddiagnostic purposes, cryptopain was cloned, sequenced and genomicSouthern analysis was performed to determine whether there was one ormore cysteine proteinase similar to cryptopain.

[0095] Degenerative oligonucleotides were synthesized from the sequencesencoding the active sites of papain like cysteine proteinases centeredaround the active site cysteine and histidine as seen in FIG. 1 andaround the active site arginine described in Example 2. In FIG. 1, theprimary structures of cysteine proteinases for L. mexicana, T. brucei,and human cathepsin-L are compared to the primary structure of C. parvumcryptopain. The diagram in FIG. 1 shows the conserved cysteine andhistidine residues involved in the active site, and the cysteineresidues apparently involved in disulfide bridges. For cryptopain, theconserved cysteine is C-24, the conserved histidine is H-164. Theproposed disulfide bridges are 21-65, 56-103 and 158-210. FIG. 1 is amodified FIG. 19.4, from Biochemical Protozoology, 214, Ed. G. Coombs,et al., Tayla and Francis, London (1991).

[0096] For fragment amplification, a number of oligonucleotides weretried without success until oligonucleotides specific for the Plasmodiumvinckei cysteine proteinase, described in Example 2 were identified.These oligonucleotides were found to be suitable for and were thereforeused to amplify a fragment of genomic DNA from Iowa isolateCrytosporidium parvum oocysts.

[0097] The fragment was sequenced using methods described below andknown in the art and found to encode a 459 bp portion of a cysteineproteinase gene seen in FIG. 2, DNA residues 869-1326. The fragment washybridized to an Iowa isolate genomic Southern blot which indicated thatthe cysteine proteinase was a single copy gene. Results are seen in FIG.5.

[0098]FIG. 5 is a genomic Southern analysis of Crytosporidium DNA usingthe cryptopain probe. In FIG. 5, lane 1, the probe hybridizes to twoHind III fragments. These fragments are of approximate size 1.5 and 4kb. In lane 2, the probe hybridizes with a Hae III fragment of 1.2 kb.In lane III the probe hybridizes to fragments of 1.2 and 4 kb of a HindIII/Hae III digest. In lane 4, the probe identifies fragments of 10 and1 kb in an NsiI digest. In lane 5, the probe identifies a single band of4 kb in an ScrII digest and in lane 6 it identifies fragments of 1.0,0.5 and 4 kb in an NsiI/ScrII digests. The presence of 1 or 2 bandsgreater than the size of the probe in all digests indicates that thecysteine proteinase is a single copy gene.

[0099] The 459 bp Iowa fragment was then used to identify naturallyinfected neonatal calf (NINC) according to Infect. Immun., 61:40 (1993)library clone which encoded the entire gene and 5′ and 3′ flankingregions. The sequence of this clone appears in FIG. 2 and is identifiedas (SEQ ID NO: 1). The sequence of the open reading frame wasdetermined.

[0100] The corresponding sequences of the NINC clone and the 459 bpsequence of the Iowa cysteine proteinase isolate are identical.indicating that cryptopain is highly conserved in these isolates andthat its function is essential for Crytosporidium.

[0101] Sequences identified as SEQ ID NOs: 1-6 disclosed in thisinvention are new. These sequences represent nucleotides and amino acidsequences of C. parvum antigen. They were prepared according to methodsdescribed in Examples 1, 2 and 3.

[0102] SEQ ID NO: 1 is the DNA sequence of the Crytosporidiumcryptopain. The sequence (SEQ ID NO: 1) comprises 1663 base pairs andcomprises 5′ and 3′ flanking sequences, pre, pro (SEQ ID NO: 2) andmature enzyme (SEQ ID NO: 3) sequences.

[0103] SEQ ID NO: 4 is the amino acid sequence of the cryptopain. Thecryptopain contains 401 amino acids and contains pre and pro fragments(SEQ ID NO: 5), and mature enzyme (SEQ ID NO: 6).

[0104] Sequences 7-8 are known and correspond to cysteine proteinasesisolated from other organisms, namely from Carica and P. vinckei.Homology between these and the current C. parvum cysteine proteinase isshown and described in FIGS. 1 and 4.

[0105] Sequences identified as SEQ ID NOs: 9-12 are primer sequences.

[0106] Sequences SEQ ID NOs: 13-15 represent amino acid fragments ofcryptopain.

[0107] Sequence SEQ ID NO: 16 represents a 1206 fragment of cryptopainDNA.

C. Structure of the Cryptopain Gene and its Encoded Protein

[0108] The function of cryptopain is highly correlated with thestructure of the protein which is determined by the correspondingsequence. In addition, regulation of the function is, at least in part,dependent upon the presence of the pro sequence.

[0109] Sequence identified as SEQ ID NO: 1 (FIG. 2) is a DNA sequence ofcryptopain. Sequence identified as SEQ ID NO: 4 (FIG. 3) is itscorresponding protein. Search of the Gene Bank and Swiss Protein Bankrevealed that these sequences were highly homologous to cathepsin L-likesequences of various organisms as seen in FIG. 4.

[0110]FIG. 4 is an amino acid alignment showing marked amino acidsimilarities of cryptopain of Crytosporidium (SEQ ID NO: 4) cysteineproteinase (papain) of Carica (SEQ ID NO: 7) and mature cysteineproteinase Plasmodium vinckei (SEQ ID NO: 8). In FIG. 4, the matureenzyme of P. vinckei and the pre pro enzymes of cryptopain and papain(Carica) are lined up.

[0111] The active site cysteine shown at site 200 is embedded in a 7amino acid fragment CGSCWAF (SEQ ID NO: 13) which is conserved in allthree enzymes and was one of the sites chosen to make degenerateoligonucleotides primers listed in FIG. 7A. There is not a high degreeof conservation of sequence between the 3 enzymes around the active sitehistidine seen at 341. However, the conserved arginine at 392 isembedded in an amino acid fragment YWL/IVRNSW (SEQ ID NO: 14) which onlydiffers by 1 amino acid in P. vinckei cysteine proteinase andcryptopain. This substitution of I and L was not engineered into thedegeneracy of the P. vinckei oligonucleotide. Nonetheless, thedegenerate oligonucleotide 782 containing sequence for VRNFW (SEQ ID NO:15) and the active site cysteine oligonucleotide 781 were specificenough to amplify the 459 bp fragment. Unlike cryptopain, the P. vinckeihas a large insertion seen in amino acids 358-386 between the conservedcysteine and arginine that were the basis for nucleotide PCR of the 459bp C. parvum fragment.

D. Production of Cryptopain Recombinant Protein

[0112] Recombinant cryptopain proteins are useful as antigens forpreparing antibodies which will inactivate cysteine proteinase andprovide antibody probes to detect the presence of the organism in theenvironmental and clinical diagnostic setting. Their recombinantproduction is therefore important.

[0113] Recombinant proteins of the invention were produced as describedin Example 5. Generally, the 1203 bp cryptopain open reading frame (ORF)is engineered for in frame expression as a thioredoxin fusion protein inthe Invitrogen vector pTrxFus, or any other suitable vector seendiagrammed in FIG. 8. This vector is used to create C-terminal fusionsto E. coli thioredoxin. There is a multiple cloning site which allows inframe fusion of foreign protein with thioredoxin. Between thethioredoxin and the foreign protein there is an enterokinase cleavagesite. Enterokinase treatment permits the release of thioredoxin from theprotein. pTrxFus DNA is digested with for example KpNI and XbaI and theintervening fragment is removed for example, by gel purification.

[0114] Primers 7B1 and 7B2 were used to amplify the pre pro enzymesequence from Iowa Crytosporidium DNA. The primer 7B1 has a KpN1 siteand the primer 7B2 has an XbaI site engineered into the 5′ end of theoligonucleotides. These enzymes are used to digest the amplified DNA sothat it could be inserted directionally and in frame into the KpnI/XbaIrestriction digested pTrxFus. Then, the vector, such as pTrxFus,containing the sequence for the pre pro enzyme, is used to transformcompetent E. coli cells. Ampicillin resistant transformants are thenanalyzed for plasmid DNA by restriction with KpNI-XbaI and by sequencefor the presence, orientation and reading frame of the gene. Clonescontaining the same gene are induced for expression of cryptopain andexpression of the fusion protein, such as for examplecryptopain-thioredoxin, at 57 kD, was analyzed by SDS-PAGE as seen inFIG. 9, followed by immunoblot with antithioredoxin antibody. Conditionsfor optimal production of soluble protein in E. coli are assessed.

[0115] Results of the actual preparation of recombinant cryptopain usingvector pTrxFus are seen in FIG. 9. FIG. 9 shows proteins harvested froma lysed cell culture, i.e., the soluble supernatant proteins. Lane 1 iswild type thioredoxin. Lanes 2, 3 and 4 are thioredoxin cryptopainsharvested from cell culture at 2, 3 and 4 hours of growth of thioredoxincryptopain. The pellet fraction showed no fusion protein indicating thatthe cryptopain-thioredoxin is wholly soluble. Growth was maximal at 3hours and degradation products of Mr less than 57 were visible at 4hours indicating that the optional time for harvesting culture wasaround 3 hours.

[0116] Fusion protein may be purified by osmotic shock or heat treatmentof cell lysates to produce highly purified fusion protein. The fusionprotein is advantageously cleaved with enterokinase at a cleavage sitecomprising 4 asparagine and 1 lysine sequence.

[0117] Production of cryptopain may be accomplished in multipleprocaryotic or eukaryotic cells, including baculovirus, insect cells,yeast and mammalian cells. Cryptopain is purified by any suitable methodknown in the art, such as incorporation of histidine and purification bynickel chromatography, heat treatment of fluoredoxin fusion protein withsubsequent harvesting of soluble protein.

I. Inhibition of Sporozoite Invasion

[0118] In order to determine whether the invasion of Crytosporidiumsporozoites may be inhibited, active site inhibitors of cathepsin L-likecysteine proteinases were investigated.

[0119] Cryptosporidiosis infection is caused by the invasion of cellswith Crytosporidium, typically Crytosporidium parvum. In order toprovide prophylactic, therapeutic, diagnostic or detection agents, it isnecessary to determine what the function of cryptopain in the process ofcell invasion is, and whether or not during the Crytosporidium cellinvasion cryptopain acts at the surface of the sporozoites. For thisreason, studies were performed using known inhibitors to determine entryof the sporozoites into the cells.

[0120] Because the sequence of cryptopain (SEQ ID NO: 4) had highhomology with other cysteine proteinases, and has an N-terminalhydrophobic region, it was decided to determine if C. parvum secretedcysteine proteinase. The biotin modification ofphe-ala-fluoromethylketone (BPAFMK) makes it unable to enter intactcells. Therefore, the biotinylated phe-ala fluoromethylketone was usedto determine whether a cathepsin-L like cysteine proteinase was activeeither at the surface of the Crytosporidium sporozoites or in thesupernatant media during invasion of Madin Darby canine kidney (MDCK)host cells and whether it allows C. parvum to enter the cells. Resultsare seen in FIG. 10.

[0121]FIG. 10A shows the % of sporozoites invasion as a function of theconcentration of three cysteine proteinase inhibitors, namely inhibitorstrans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E64), obtainedfrom Sigma, St. Louis, BPAFMK, obtained from Enzyme Systems, Dublin,Calif. and K-III, research drug of Arris Pharmaceutical, South SanFrancisco, Calif. The inhibitors were administered within the range of10⁻³ to 10¹ (0.001 to 10 mM).

[0122] As seen in FIG. 10, one hundred nM of all three inhibitorsdecreased invasion by Crytosporidium to 20-30% of untreated controls.The inhibition of invasion of sporozoites by BPAFMK (FIG. 10) shows thatcryptopain is involved in a proteolytic events which are necessary forinvasion and intracellular development of Crytosporidium. Results seenin FIG. 10 therefore show that cryptopain is either localized at thesurface of the Crytosporidium sporozoites, is a part of the sporozoitesmembrane or is localized internally and is released during the invasionof the host cell.

[0123] Assessment of other studied cysteine proteinase inhibitors (E64and K-III) which were not chemically modified to prevent entry into thecell indicate that there is more than one cathepsin-L-like cysteineproteinase inhibitor which will prevent invasion and intracellulardevelopment.

[0124] Although not listed here, it is to be understood that othercysteine proteinase inhibitors, as long as these inhibitors inhibitCrytosporidium invasion, are intended to be within the scope of thisinvention. The examples of active site inhibitors aretrans-epoxysuccinyl-L-leucylamido-(4-quanidino) butane (E64),fluoromethylketone, diazomethanes, vinyl sulfones and cystatins.

[0125] Another class of inhibitors derived from pro region of cryptopainand its derivatives change the active enzyme into a proenzyme.

[0126] As described above, the complete DNA and amino acids structuresof cryptopain (SEQ ID NOs: 1 and 4) comprise pre, pro and mature enzyme(SEQ ID Nos: 2 and 5) sequences (SEQ ID NOs: 3 and 6), delineated inFIG. 1 and in FIG. 4. These pre, pro and mature regions or elements areidentified on the basis of homology to previously discovered andinvestigated cysteine proteinases, seen in FIG. 1, compared toCrytosporidium parvum cryptopain. Biochemical Protozoology, (supra). TheN-terminus of the cryptopain sequence contains pre pro sequence (SEQ IDNO: 2). The cryptopain pre sequence is predicted by Kyto-Doolittlehydropathy plot seen in FIG. 6 to contain a hydrophobic sequence. Suchsequences typically target the protein to a membrane.

[0127] Inactive cysteine proteinases are called proenzymes. Proenzymesof cysteine proteinases consist of at least a pro amino acid sequencewhich interacts conformationally with the contiguous mature enzymesequence to render it inactive until the pro sequence is cleavedreleasing the active mature enzyme. Recent evidence indicates that thepro sequences of cysteine proteinases are excellent specific inhibitorsof their respective mature active enzymes (Protein Eng., 8:59 (1995)).

[0128] Thus, the pro sequence (SEQ ID NO: 5) of cryptopain is a goodcandidate and may be produced by recombinant or synthetic means for useas a pharmacological agent to prevent Crytosporidium infection and/orthe consequences of infection.

III. Prophylaxis or Treatment via Passive or Active Immunization

[0129] For protection and treatment of human or animal subjectssubjected to exposure to Crytosporidium, or subjects already sufferingfrom Crytosporidium infection, both passive or active immunization usingthe cryptopain antigen is appropriate.

[0130] Surface active enzymes with confirmed essential functions for theparasite infectivity, like cryptopain, are targets for passive or activeimmunization. Cryptopain binds to antibodies which specifically bind toepitopes of Crytosporidium which are recognized by B and T cells.

[0131] For prophylactic, therapeutic or diagnostic purposes, theproteins of the invention are produced in large amounts by inserting theCrytosporidium parvum DNA, described above, into an expression vectorsuch as pGEX, pET-9d, pTrxFus or baculovirus obtained from Invitrogen.The thus constructed hybrid vector is then used to transform ortransfect a host. The host cells carrying the hybrid vector are thengrown in a nutrient medium to allow the production of the gene product.

[0132] A number of transfer vectors are available for the production ofprotein from both full length and partial cDNA and genomic clones. Fusedor non-fused protein products, depending on the vector used, constituteup to 50% of the total protein produced in infected cells. The thusobtained recombinant proteins are frequently immunologically andfunctionally similar to the corresponding endogenous proteins.

[0133] The obtained polypeptide is purified by methods known in the artor described in Examples. The degree of purification varies depending onthe use of the polypeptide. For use in eliciting polyclonal antibodies,the degree of purity may not need to be very high. However, as in somecases impurities may cause adverse reactions, purity of 90-95% istypically preferred and in some instances even required. For thepreparation of a pharmaceutical composition, however, the degree ofpurity must be high, as is known in the art.

[0134] When in a therapeutic composition, the polypeptide is combinedwith appropriate pharmaceutically acceptable excipients adjuvants andused for the immunization of immunocompetent patients who are at riskfor cryptosporidiosis either at the time of immunization or in thefuture.

[0135] This group includes, but is not restricted to, HIV positiveindividuals who are still able to respond to vaccination, animalworkers, health care workers, day care center children and theircaretakers, and children in the developing world.

A. Antibodies and Their Production

[0136] A polyclonal or monoclonal antibody to native or recombinantprotein of the invention are useful in diagnosing and detectingCrytosporidium as well as for treatment by providing a protectionagainst the Crytosporidium infections.

[0137] Anti-Crytosporidium polyclonal antibodies recognizing the clonedpolypeptide are preferred over a monoclonal antibody (MAb) because theyrecognize multiple epitopes on the target polypeptide.

[0138] According to the method of the current invention, large amountsof recombinant cryptopain are produced by scale up processes incommercial plants which enables production 9f a corresponding largequantity of polyclonal antibodies or of immunogen for activeimmunization. The antibodies to recombinant expressed protein can alsobe produced according to the invention using the standard methodavailable for production of the antibodies to native protein.

[0139] Cryptopain comprising epitopes of Crytosporidium that isrecognized by intact B and/or T cells is produced in large amounts asdescribed above and in Examples, purified and used to detect orcharacterize anti-Crytosporidium parvum antibody in the body substancesof populations at risk of prior or current cryptosporidial infection.Cryptopain is also used for immunization. Typical intramuscularimmunization schedules are as follows.

[0140] Cryptopain plus equal volume complete pharmaceutically acceptableadjuvants and excipients is used at the beginning of immunization.Antigen plus equal volume incomplete adjuvant is used at week 2. Antigenplus equal volume incomplete adjuvant at week 4.

[0141] In addition, antibodies to such antigens are obtained byimmunizing animals, such as rabbits or goats, with the polypeptide plusadjuvant, as described above.

[0142] The antibodies of the invention are also used to detectCrytosporidium antigens in body substances, for example, stools ofpopulations at risk of cryptosporidial infection by, e.g., collectingstool samples (Manual of Clinical Microbiology, 1986, supra), mixingwith Streather's solution 1:4, and incubating with antibody followed byaddition of a fluorescein conjugated second antibody. In alternative,colorimetric labeling which do not require special microscope equipmentor other detection methods also suitable.

B. Biologically Derived or Recombinant Anti-Cryotosporidium Vaccines

[0143] Vaccine is a biologically derived or recombinantly prepared agentuseful for artificially acquired immunization in a host. The currentinvention describes a production and provides biologically derived andrecombinant vaccines for active immunization of animals and humansagainst cryptosporidiosis and for the preparation of passive immuneproducts for treatment of the established infection.

[0144] The scope of the invention is, therefore, intended to includeboth biologically derived or recombinantly prepared vaccines based onthe antigens of the invention.

[0145] A recombinant vaccine is produced by identifying the relevantantigen or antigens of Crytosporidium species, cloning them andexpressing them using suitable vectors. This approach yields immunogenswhich are reproducible in sufficiently large quantities to allowpreparation of vaccine for active immunization. Recombinant vaccines areuseful for immunization of the potential Crytosporidium host, such asfor example for inoculation of cows, and to produce the host's ownantibodies against Crytosporidium infection.

[0146] Additionally, the recombinant vaccines may be used for productionof passive immunotherapeutic agents. For example, when the cow isinoculated with the vaccine it begins to produce hyperimmune colostrum.Hyperimmune colostrum from the immunized cows is then purified to yieldIg for passive immunotherapy of immunocompromised persons, primarilyAIDS patients, children, etc.

[0147] These vaccines are also useful for widespread use in calves toprovide a primary protection against Crytosporidium infection. Providingthe herd with anti-Crytosporidium immunity decreases the risk forwaterborne outbreaks of cryptosporidiosis in areas where the watershedincludes dairy lands. This provides a secondary benefit to humanresidents of those areas.

[0148] In addition, DNA or RNA may be introduced into a host such thatpropagation and/or expression of the encoded protein occurs in the hostutilizing a so called “foreign expression system”.

[0149] Anti-Crytosporidium vaccine of the invention contains aCrytosporidium antigen identified by the invention, modified in such away that it is incapable of producing the Crytosporidium symptoms but atthe same time it is capable of eliciting the production of specificprotective antibodies against the disease when introduced in the body.

[0150] Protection from cryptosporidiosis appears to be due to mucosalimmunity which, if absent in AIDS patients, is difficult to establishbut, if present, may afford protection against clinicalcryptosporidiosis as AIDS progresses.

[0151] Thus, the invention describes vaccines able to provide active Bcell-immunity and potentially T cell immunity against cryptosporidiosisin normal persons, in persons at risk for AIDS or in otherwiseimmunocompromised patients.

C. DNA and RNA Vaccines

[0152] Recently, new approaches appeared which utilize so called DNA orRNA vaccines. These approaches are described in Science, 259:1745(1993), hereby incorporated by reference.

[0153] DNA or RNA vaccines or native immunity are produced according tothe methods described Ibid. Briefly, nucleic acid vectors containingCrytosporidium antigen DNA nucleic acid are injected, preferablyintramuscularly to the host. The nucleic acid enters or is transmittedwhere it results in production of antigen. The antigen elicits immuneresponses in the form of specific anti-Crytosporidium antigen antibodyor cell medicated immune events. In this way, the host receives DNA orRNA and provides his/her own humoral immunity and/or cell mediatedresponses.

IV. Diagnostic/Detection Utility

[0154] An important part of this invention is a method of diagnosingCrytosporidium infection or detection of Crytosporidium in the tissuesamples or in the environment.

[0155] The diagnostic method comprises contacting a body fluid or tissuewith an anti-Crytosporidium polyclonal or monoclonal antibody havingspecificity for the antigen of this invention or its fragments, or viceversa, and ability to detect any selective binding of the antibody toany antigenic Crytosporidium proteins present in the body fluid, tissueor specimen or selective binding of the antigen to theanti-Crytosporidium antibody.

[0156] The detection of the antibody-antigen complex in body specimensor environmental samples may be conducted by any method known in theart. The detection methods include solid phase, double antibody,sandwich double antibody, and triple antibody assays, including ELISAand the like. Also suitable are enzyme-linked immunoassays andradioactively labeled assays.

[0157] Examples of body specimens are stools and other liquid or solidbody output or tissue samples obtained from a subject. Examples of bodyfluids are blood, serum, saliva, urine, and the like. Methods for thepreparation of the body substance and the body fluid are standard in theart and are described, for example in Manual of Clinical Microbiology,Chapter 8, “Collection, Handling and Processing of Specimens”, 4thedition, Eds, Lennette, E. H., Balows, A., Hausler, W. J. and Shadorny,A. J., American Society for Microbiology, (1986)).

[0158] Diagnosis and detection methods also comprise contacting the DNAand RNA of body fluid, tissue, specimen and environmental sample withDNA and RNA of the invention or fragments thereof and the amplificationof this specific interaction via PCR, branched chain nucleic acidtechnology and other amplification technologies such that the presenceof Crytosporidium DNA and/or RNA in the bodily fluid, tissue, specimenor environmental sample may be detected. Agents suitable forimmunodiagnostic use are proteins comprising epitopes of Crytosporidiumparvum that are recognized by intact B and/or T cells. These proteinsare produced as described above, purified and used to detect orcharacterize anti-Crytosporidium parvum antibody in the body substancesof populations at risk of prior or current cryptosporidial infection. Inaddition, antibodies to such proteins are obtained by immunizinganimals, such as cows, rabbits or goats, or birds with the vaccinecombined with an adjuvant.

[0159] Additionally, detections of Crytosporidium may be made bydetermining cryptopain activity in biological or environmental samplesby methods used and known in the art.

V. Immunotherapy and Prophylaxis

[0160] The immunotherapy of cryptosporidiosis in humans and animals maybe conducted by administration of the antibodies of the invention topatients with cryptosporidiosis to effectively reduce theirsymptomatology.

[0161] A method for immunotherapeutic treatment, retardation, orinhibition of Crytosporidium infection comprises administering to asubject in need of such treatment an amount of an anti-Crytosporidiumpolyclonal or monoclonal antibody prepared according to the invention,effective to provide immunity against the invasion of Crytosporidium oreffective to inhibit the existing Crytosporidium infection.

[0162] A method of prophylaxis of Crytosporidium infection comprisesadministering to a subject in need of such treatment a vaccine, asdescribed above, comprising the protein or recombinant protein of thisinvention capable of endogenous development of inhibitory amount ofanti-Crytosporidium parvum antibodies.

[0163] Typical immunization is achieved by inoculation of the animal,bird or human host with the antigen protein combined with adjuvant.

[0164] For passive immunotherapy when used to passively immunizeCrytosporidium infected hosts, the polypeptide is first combined withappropriate adjuvants and used for the immunization of cows or otherdonor animals to produce antibodies which may be administered topatients with cryptosporidiosis infection, particularly to AIDSpatients, and to other immunocompromised hosts. Monoclonal antibodiesproduced in animals, in humans “humanized” from animal sources andproduced through chimeric techniques and other derivative techniques maybe used for passive immunotherapy.

[0165] When in a therapeutic composition, the antigen protein iscombined with appropriate adjuvants and used for the immunization ofimmunocompetent patients who are at risk for cryptosporidiosis either atthe time of immunization of in the future. This group includes, but isnot restricted to, HIV positive individuals who are still able torespond to vaccination, animal workers, health care workers, day carecenter children and their caretakers, and children in the developingworld.

VI. Qualitative and Quantitative Detection ofCryptosporidium-Formulations and Kits

[0166] Formulations suitable for the administration of polypeptides andantibodies such as those described herein are known in the art.Typically, other components stimulatory of immune response as well asfillers, coloring, and the like may be added, such as pharmaceuticallyacceptable excipient, additives and adjuvants.

[0167] For qualitative and quantitative determination of the presence ofthe Crytosporidium infection and environmental contamination, a kit forthe diagnosis/detection of Crytosporidium is used. The kit comprises thepolyclonal antibody or antigen of this invention and a means fordetecting the complexing of the antibody with antigen. Another such kitcomprises DNA/RNA of the invention for use in detecting complementaryDNA/RNA of cryptopain. Another such kit comprises PCR primers foramplification of cryptopain sequences and a method of identifying them.

[0168] The kit is utilized for the detection of endogenousantibodies/antigens/DNA/RNA produced by a subject that is afflicted withcryptosporidiosis and antigens/DNA/RNA present in the environmentalsamples. Even at the early stages where the parasite is commencinginvasion of a subject's cells, some amount of the Crytosporidium antigenor the specific antibody may be detected in serum. The kit detectseither the antigen with the polyclonal antibodies or the presence of theanti-Crytosporidium antibody with the antigen. The complexingimmunoreaction is detected by staining, radiography, immunoprecipitationor by any other means used in the art and suitable for these purposes.

[0169] In addition to the above, the kits may also comprise a controlcompounds, anti-antibodies, protein A/G, and the like, suitable forconducting the different assays referred to above.

[0170] The current invention provides an effective treatment andprophylaxis against the cryptosporidiosis infection and means ofdetection of the parasite and diagnosis of infection.

[0171] The following examples describe procedures used to prepareantigens, antibodies, vaccines and kits of the invention. They areillustrative only and any modification using methods known in the art isintended to be included. The following examples are not to be consideredin any way limiting.

EXAMPLE 1 Cryptosporidium parvum Parasites

[0172] This example describes protocol used for isolation ofCrytosporidium parvum parasites from which the Crytosporidium antigenwas prepared.

[0173] Oocysts of the Iowa isolate of Crytosporidium parvum werepassaged through neonatal calves, (Pat Madin Pathasan, Pleasant HillFarms, Idaho). The passaged oocysts were purified and encysted for usein invasion assays. The detailed protocol for purification and encystingis described in Infect. Immun., 61:4079 (1993). The described protocolwas used unmodified.

[0174] For the DNA experiments described herein DNA was purified from1×10⁹ Crytosporidium parvum according to Mol. Biochen Parasitol,50:105-114, (1992).

EXAMPLE 2 Preparation of a C. parvum Cysteine Proteinase DNA Probe

[0175] This example describes procedural used for preparation of the C.parvum cysteine proteinase DNA probe.

[0176] Cysteine proteinases share DNA homology in the regions coding forthe active site amino acids involved in proteolysis, specificallyconserved C, H and N residues. This was used in choosing an appropriateoglionucleotide pairs to amplify the genomic DNA from Iowa isolate. Themost suitable oligonucleotides were found to be those modeled inPlasmodium vinckei cysteine proteinase sequences around the conservedcysteine (C) and arginine (N) residues as indicated in FIG. 4.

[0177] Degenerate oligonucleotides for the active C and N sites of thePlasmodium vinckei cysteine proteinase were used to amplify a 459 bpgenomic DNA fragment from Iowa isolate DNA. In the degenerateoligonucleotides a “/” indicates that the base pair on either side ofthe “/” could be included at that location in a triplet encoding anamino acid. (I) indicates inosine which will pair in hybridizationreactions in a permissive manner. The oligonucleotides were PC4(sense)consisting of AAA-GGA-TCC-TGC/T-GGI-A/TG/CI-TGC/T-TGG-GCI-TT (SEQ ID NO:9) encoding a BamHI site and the DNA sequence for C-G-S-C-W-A-F (SEQ IDNO: 13) and PC3 (anti-sense) consisting of the DNA sequenceTTT-GAA-TTC-CCA-IG/CA/T-A/GTT-IC/TT/G-IAC/T-IAT-CCA-A/GTA (SEQ ID NO:10) encoding an Eco RI site and the antisense for a protein sequence.The protein sequence in the sense direction is Y-W-I-V/I-K/R-N-S-W (SEQID NO: 14). The restriction sites were not required for the experimentsdescribed here.

[0178] As shown in FIG. 4, these oligonucleotides represented a 100%match for the seven amino acid C-G-S-C-W-A-F (SEQ ID NO: 13) sequence ofC. parvum cryptopain and a 100% match for the five amino acids V-R-N-S-W(SEQ ID NO: 15) within the eight amino acid sequence surrounding theconserved N. These matches were sufficient for PCR amplificationpurposes.

[0179] One hundred nanograms of Iowa isolate DNA was amplified usingreagents from GeneAmp (Perkin-Elmer, Foster City, Calif.) under thefollowing conditions:

[0180] Initial denaturation was 94° C. for 2 minutes followed by 30cycles of 94° C. for 20 seconds, 40° C. for 40 seconds and 72° C. for 1minute.

[0181] The 459 bp amplification product was isolated, subcloned in theTA vector (TA Cloning kit, Invitrogen, San Diego, Calif.) and sequencedusing the di-dioxy technique (Stategene Sequenase II Kit).

[0182] Iowa isolate sequence so obtained was found to be homologous tocysteine proteinases of a wide variety of organisms. The sequences ofpapain and the cysteine proteinase of P. vinckei are shown in FIG. 4.

EXAMPLE 3 Isolation, Sequencing and Analysis of a C. parvum CysteineProteinase Gene

[0183] This example describes isolation, sequencing and analysis of a C.parvum cysteine proteinase gene encoding C. parvum antigen.

[0184] The 459 bp amplification product obtained in Example 2,containing a portion of an Iowa isolate C. parvum cysteine proteinasegene was labeled with d-dATP³² using random primers and Klenow fragment.The labeled 459 bp probe was used to screen a NINC, (naturally infectedneonatal calf) λgt11 genomic expression library.

[0185] Three clones, designated E1.6, E4 and RCB1.2, were identified inthe library and were purified to homogeneity. Two of them, E1.6 and E4,were subcloned in Bluescript for sequencing (Sequence II kit) and werefound to contain the complete cryptopain sequence and 5′ and 3′ flankingsequences as determined by analysis of the open reading frames withinthe clones and Genebank Search using the deduced amino acid sequence.

[0186] The entire sequence of E1.6 is designated SEQ ID NO: 1 andincludes flanking sequences 5′ and 3′. The mature enzyme sequence isdesignated SEQ ID NO: 3.

EXAMPLE 4 Southern Hybridization

[0187] This example describes Southern hybridization method used todetect the gene of the invention in genomic DNA.

[0188] One mg of Iowa DNA was digested with the restriction enzymes,Hind III, Hae III, Nsi, Scr II and combinations thereof, according toconditions for use of each enzyme as provided by the manufacturer(Promega). Digested DNAs were subjected to electrophoresis in 0.8%agarose gels in 1×TAE. The gel was blotted to a nylon membrane HybondN+, obtained from Amersham per manufacturer's instructions.

[0189] Results are seen in FIG. 5 which shows a generic Southernanalysis using DNA cut and separated in this manner (lane 1=Hind III,lane 2=HaeIII, lane 3=HindIII/HaeIII, lane 4=NsiI, lane 5=SrcII and lane6=Nsi/SrcII). The 459 bp probe was labeled with ³²P-ATP and hybridizedto the membrane.

EXAMPLE 5 Preparation of Recombinant Cryptopain

[0190] This example describes the preparation of recombinant cryptopain.

[0191] The primers 7Bl and 7B2 (FIG. 7B) were synthesized at theBiomedical Research center, University of California, San Francisco. 7Blis a sense oligonucleotide and is comprised of a KpnI restriction enzymerecognition site at the 5′ end followed by coding sequence for the 5′end of the pre pro cryptopain sequence. 7B2 is an anti-senseoligonucleotide and is comprised of an Xbal sequence at the 5′ endfollowed by the antisense coding sequence of the 3′ end of thepreprocryptopain sequence. When used as a pair of PCR amplificationoligonucleotides, these oligonucleotides allowed the amplification fromgenomic Crytosporidium DNA of the entire cryptopain gene with Kpn 1 andXba I sequences at the 5′ and 3′ ends respectively.

[0192] The 7BI and 7B2 sequences were designed so that after Ypnl andXba I digestion of the amplification product, the resultant fragmentcould be introduced in a directional manner into pTrxFus which was cutwith Kpnl/XbaI. Amplified and restricted DNA was visualized on a 0.8%agarose-1XTAE gel using ethidium bromide. The amplified and endonucleaserestricted band was excised from the gel and purified using a glass beadtechnique (Gene-Clean).

[0193] pTrxFus was also digested with the enzymes KpnI and Xba I,enzymes uniquely present in the sequence in the poly linker (FIG. 8),and the small intervening sequence was removed by gel purification asnoted above. pTrxFus and preprocryptopain DNA, prepared in this manner,at 1:1 and 1:5 molar ratios were ligated overnight at 14° C. in thepresence of ligation buffer and T4 DNA ligase at a concentration of50-250 ng insert DNA/10 μl.

[0194] G1724 chemically competent cells were made as described byXi-Lvitrogen. Three to five μl of ligation mixes and control mixes wereintroduced into separate tubes of competent cells and the tubes wereincubated on ice for 30 minutes. Tubes were incubated in a 42° C.heating block for 90 seconds and placed on ice for 2 minutes. Eighthundred μl of room temperature of enriched tryptone containing brothmedium was added to each tube and the tube was incubated with shaking at30° C. for 60 minutes. Twenty-five and 100 μl of each transformation mixwas plated on RMG-Ampicillin transformation plates and the plates wereincubated at 30° C. overnight.

[0195] Nitrocellulose membrane replicas of colonies were prepared fromthe transformation plates, the adherent cells lysed in alkaline solutionand the DNA fixed to the membranes. Nitrocellulose membranes werehybridized with probes to contain cryptopain DNA and followinghybridization with a cryptopain, probe were colony purified. DNA waspurified from colonies and the identity of the foreign DNA verified byrestriction analysis and sequence analysis.

[0196] Purified colonies were grown in 1 μl aliquots for analysis.Growth conditions were varied with respect to time (2, 3, 4 hours) andthe bacteria lysed for evaluation of soluble and insoluble proteins.Results are shown in FIG. 9.

[0197]FIG. 9 shows soluble proteins from 10 μl of lysate at 2, 3, 4hours in lanes 2, 3, and 4 on an immunoblot of an SDS-PAGE gel.Cryptopain fused to thioredoxin appears as a 57 kDa protein which isappropriate for the size of the fusion partner (12 kDa) and the size ofpreprocryptopain (35 kDa). Lane 1 is the thioredoxin control. All lanesare visualized with anti-thioredoxin antibody followed bychemiluminescent detection (Amersham). Yield, using this expressionsystem, was maximal at 3 hours of bacterial growth and was estimated at0.9 mg cryptopain-thioredoxin per 250 ml culture. Although the yield wasvery high in this system, purification after enterokinase removal of thefusion partner was less satisfactory.

EXAMPLE 6 Large Scale Purification of Recombinant Cryptopain

[0198] This example describes the purification procedure for cryptopain.

[0199] In order to provide large quantities of cryptopain purified fromits fusion partner, thioredoxin, the KpnI/Xbal preprocryptopain DNAfragment of Example 5 was cloned into an improved vector known as pThioHis (Invitrogen). The improvements of the invitrogen system were:

[0200] 1) Metal binding sites were engineered into the sequence betweenthe thioredoxin reading frame and the enterokinase recognition sitefacilitating large scale purification of the fusion protein overchromatography columns (Pro-bond, Invitrogen). 2) Growth of transformedbacteria (Top 10, Invitrogen) in the presence of more standard media. 3)Ability to cleave the foreign protein from the fusion partner usingenterokinase while the fusion protein was on the nickel column allowinga high degree of purification from the fusion partner.

[0201] Colonies were prepared as in Example 5 using Top 10 E. coli.Large scale group was accomplished, the bacteria harvested and lysed andthe fusion protein collected by passage over Probond or other metalchelation columns. The columns were washed with normal saline andcryptopain was collected by passing dilute enterokinase over the column.

EXAMPLE 7 Inhibition of Cryptosporidium Invasion and IntracellularDevelopment in MDCK cells with Inhibitors of Cathepsin-L Like CysteineProteinases

[0202] This example describes studies performed to detect inhibition ofCrytosporidium invasion and intracellular development in vitro usingcathepsin-L-cysteine proteinase inhibitors.

[0203]Crytosporidium oocysts of the Iowa isolate were encysted accordingto Example 1. To assess the effect of inhibitors E64, BPAFMK and KIII onsporozoite invasion, inhibitors were incubated with viable sporozoitesfor 30 minutes prior to addition to monolayers of MDCK cells asdescribed in (J. Protozool., 386:556 (1991); and Infect. Immunol.,61:4079 (1993).

[0204] Sporozoite invasion and intracellular development in MDCK cellswas scored at 16 hours after fixation of MDCK cells in formalin andstaining with Giemsa.

EXAMPLE 8 Detection of Proteinase Activity as a Measure of Viability ofCryptosporidium Organisms

[0205] This example describes a method for detection of proteinaseactivity as a measure for viability of Crytosporidium organism inenvironmental samples.

[0206]Crytosporidium cannot be grown in culture in vitro. Availableevidence indicates that acquisition of cryptosporidiosis from water,food and other environmental sites is a major source of disease spread.However, reliable methods of determining whether living Crytosporidiumspecies are present in a sample have not been developed.

[0207] The invention provides a method assaying activity of proteinswhich have a short half-life. Proteinases which are tightly regulatedwith respect to activation, because unrestricted activity would damagethe integrity of the cell, represent one such type of proteins.

[0208] Highly specific active site inhibitors of cryptopain are used forevaluation of viability of Crytosporidium organisms. A highly specificinhibitor of cryptopain, for example E64, KIII or pre pro cryptopainprotein is labeled with a radioactive, chemiluminescent, colorimetric orother tag. The tagged inhibitor is incubated with Crytosporidiumorganisms/proteins from an environmental sample and the amount of tagbound/organism relative to positive and negative control is ascertained.Number of organisms may be determined by flow cytometry.

EXAMPLE 9 Agents Suitable for Passive Immunotherapy

[0209] This example describes preparation of suitable agents for passiveimmuno therapy.

[0210] Recombinant cryptopain described in Example 5, or a recombinantfragment of cryptopain with or without fusion protein are used toimmunize animals such as cows, goats or rabbits. The antibody developedin the body of the animal is purified from serum or milk as colostrum orused without purification for treatment of a Crytosporidium infection ofmucosal surfaces.

[0211] The antibody is delivered orally or through a tube and isoptionally mixed with agents or substances which delay or prevent theinactivation of antibody in the gastrointestinal tract.

EXAMPLE 10 Agents Suitable for Active Immunotherapy

[0212] This example illustrates agents derived from C. parvum suitablefor active immunotherapy.

[0213] Recombinant cryptopain according to Example 5, or recombinantfragments of cryptopain with or without fusion protein is used toimmunize animals or humans in such a way that the animal or humandevelops antibody or cell mediated immune responses to Crytosporidiumwhich ameliorate or inhibit infection by Crytosporidium.

EXAMPLE 11

[0214] Agents Suitable for Immunodiagnostic/Immunodetection Use

[0215] This example illustrates procedure for obtaining agents derivedfrom Crytosporidium parvum for suitable immunodiagnostic/immunodetectionuse.

[0216] Recombinant cryptopain or recombinant fragments of cryptopain orantibodies (monoclonal, polyclonal or chimeric) raised to recombinantcryptopain or recombinant fragments of cryptopain are used to detect thecorresponding antibody or antigen in a soluble or fixed assay.

[0217] Recombinant cryptopain is immobilized in wells and utilized todetect the corresponding antibody from humans or animals by capture ofthe antibody and colorimetric or other detection method.

[0218] Correspondingly. antibodies to recombinant cryptopain areimmobilized in wells and utilized to detect cryptopain in secretions orfeces or other bodily fluids or environmental samples. Both of theseassays are also be performed in a soluble format.

EXAMPLE 12 Detection of MRNA as a Measure of Viability ofCryptosporidium Organisms

[0219] This example illustrates detection of mRNA as a measure ofviability of Crytosporidium organisms.

[0220] The presence of mRNAs which have a short half-life was assayed onthe basis of the fact that many mRNAs are destroyed within 2 minutes ofproduction and the amount of intact MRNA in a cell provides a measure ofthe viability of an organism.

[0221] A probe for hybridization with the MRNA of the invention isprepared and labelled with radioactive, chemiluminescent, colorimetricor other tag. The tagged probe is incubated with Crytosporidiumorganisms from an environmental sample and the amount of tag bound/cellrelative to positive and negative controls is ascertained. Number oforganisms is determined by flow cytometry or any other suitable means.

EXAMPLE 13 Agents Suitable For Nucleotide Based Diagnosis/Detection Thisexample illustrates the procedure for obtainin agents derived from C.parvum for nucleotides based diagnosi and/or detection.

[0222] Oligonucleotides or PCR amplification products using nucleotidesderived from the cryptopain or the flanking DNA sequences is used todetect Crytosporidium in human or animal samples or in the environment.

[0223] Oligonucleotides are used to amplify a Crytosporidium fragment asdescribed in the Examples above from the samples or from the environmentand to detect its presence in either location. PCR amplificationproducts or segments of DNA or RNA are used as probes to detect thepresence in either location in hybridization experiments. Hybridizationis either as a Southern blot or as a dot blot. The hybridization signalis amplified by a variety of techniques including the branched chaintechnique.

EXAMPLE 14 Preparation of Anti-Crypotosporidium Vaccines This exampledescribes preparation of anti-Crytosporidium vaccines using DNA, RNA oramino acid cryptopain sequences.

[0224] A vaccine for prevention and treatment of infections caused byprotozoan Crytosporidium species (Crytosporidium) in humans and othermammals was developed by utilizing newly identified and isolated DNA andamino acid sequences of the Crytosporidium pathogen designatedcryptopain.

[0225] The antigen proteins used for preparation of vaccines correspondto cryptopain (SEQ ID NO: 4) which is identified by being a target ofthe polyclonal or monoclonal antibodies of the invention capable ofpreventing or ameliorating disease and preventing invasion and/orintracellular development in host cells.

[0226] A DNA or RNA vaccine for prevention and treatment of infectionscaused by protozoan Crytosporidium species (Crytosporidium) in humansand other mammals was developed by utilizing newly identified andisolated DNA (SEQ ID NOs: 1-3) and amino acid sequences of theCrytosporidium pathogen designated cryptopain.

[0227] A hybrid vector comprising a DNA segment that encodes the proteinantigen able to bind selectively and specifically to anti-Crytosporidiumantibodies operatively coupled to the vector was prepared and expressedas described in Example 5. This includes preparation of recombinantvaccines using the viral expression vector according to Example 5outside of the host body but also includes preparation of DNA vaccinesand procaryotic or eukaryotic host carrying the hybrid vector which maybe introduced into the host vertebrate or a direct introduction of DNAor RNA into host cells generating the hosts own expression ortranslation of DNA or RNA to produce proteins eliciting appropriateantibodies.

EXAMPLE 15 Preparation of Anti-Cryptosporidium parvum Vaccine

[0228] This example illustrates procedure for preparation ofanti-Crytosporidium parvum vaccine of the invention and its use.

[0229] Vaccines use of recombinant Crytosporidium antigens preparedaccording to Examples 5 and 14.

[0230] (1) Antigens

[0231] Preferably 10-200 μg of recombinant antigen of the invention,either alone or in combination is sued for preparation of the vaccine.

[0232] (2) Adjuvant

[0233] Any one of a number of adjuvants designed to either:

[0234] (a) stimulate mucosal immunity; or

[0235] (b) target mucosal lymphoid tissue is sued for preparation of thevaccine of the invention.

[0236] Examples of these adjuvants are: liposomes, saponins, lectins,cholera toxin B subunit, E. coli labile toxin (LT) B subunit, pluronicblock copolymers, hydroxyapatite, plant glucans, acetyl mannan (fromAloe Vera), aluminum hydroxide.

[0237] (3) Route of Administration

[0238] Since the vaccine must stimulate mucosal immunity, it preferablyis delivered to a mucosal site.

[0239] Feasible routes of administration include: oral, nasal, rectal,and vaginal. However, boosting may occur via another route.

[0240] (4) Volume

[0241] The volume of the vaccine, while not particularly important,should be in the range that would permit ease of use. Preferred rangewould be about 0.5 ml-2.5 ml, including adjuvant, per one vaccine dose.

[0242] (5) Boost Schedule

[0243] Since this vaccine would be intended for immunocompromisedindividuals, one would expect the diminishing immune status to require amore aggressive boosting schedule than would otherwise be necessary.

[0244] The vaccine is administered to high risk patients initially whentheir immune status is reasonably good (i.e., CD4 count of >500).Booster schedules are typically given initially at 1 month after theprimary immunization, and again every 3-4 months during progression ofthe immunodeficient state.

1 16 1663 base pairs nucleic acid double linear DNA Cryptosporidiumparvum 1 CAAAACTTCC TAATTTCTCA ATGTATTACT AATTAATAGA AAGTTTGTTTTATTTTCATG 60 TGGATAAATG AATTATTTTC TCTATACCGG CATTTGCATG CAATTTTGTATGACTAAAAT 120 GTAAATAATT ATTTGCATGC AATTATGTGG GCATGTCATA GTTTTTCAAGAATAATAATA 180 AGATGACATG ACAAGATATT CAAAAAAATT TGATGATTAT ATGTTGAAGTTAATTGAACT 240 AAAAAGTAAT TAAGTAAAAT GGACATAGGA AACAACGTGG AAGAACATCAGGAATATATT 300 TCTGGACCAT ACATTGCATT AATTAATGGC ACTAATCAAC AAAGGGAACCGAATAAAAAG 360 TTGAAAAACA TAATAATTGC AACGTTGATT GCAATCTTTA TAGTTTTGGTTGTTACTGTA 420 TCTTTGTATA TTACTAATAA CACCAGTGAC AAAATTGACG ATTTCGTACCTGGTGATTAT 480 GTTGATCCAG CAACTAGGGA GTATAGAAAG AGTTTTGAGG AGTTCAAAAAGAAATACCAC 540 AAAGTATATA GCTCTATGGA GGAGGAAAAT CAAAGATTTG AAATTTATAAGCAAAATATG 600 AACTTTATTA AAACAACAAA TAGCCAAGGA TTCAGTTATG TGTTAGAAATGAATGAATTT 660 GGTGATTTGT CGAAAGAAGA GTTTATGGCA AGATTCACAG GATATATAAAAGATTCCAAA 720 GATGATGAAA GGGTATTTAA GTCAAGTAGA GTCTCAGCAA GCGAATCAGAAGAGGAATTT 780 GTTCCCCCAA ATTCTATTAA TTGGGTGGAA GCTGGATGCG TGAACCCAATAAGAAATCAA 840 AAGAATTGTG GGTCATGTTG GGCTTTCTCT GCTGTTGCAG CTTTGGAGGGAGCAACGTGT 900 GCTCAAACAA ACCGAGGATT ACCAAGCTTG AGTGAACAGC AATTTGTTGATTGCAGTAAA 960 CAAAATGGCA ACTTTGGATG TGATGGAGGA ACAATGGGAT TGGCTTTTCAGTATGCAATT 1020 AAGAACAAAT ATTTATGTAC TAATGATGAT TACCCTTACT TTGCTGAGGAAAAAACATGT 1080 ATGGATTCAT TTTGCGAGAA TTATATAGAG ATTCCTGTAA AAGCCTACAAATATGTATTT 1140 CCGAGAAATA TTAATGCATT AAAGACTGCT TTGGCTAAGT ATGGACCAATTTCAGTTGCA 1200 ATTCAGGCCG ATCAAACCCC TTTCCAGTTT TATAAAAGTG GAGTATTCGATGCTCCTTGT 1260 GGAACCAAGG TTAATCATGG AGTTGTTCTA GTTGAATATG ATATGGATGAAGATACTAAT 1320 AAAGAATATT GGCTAGTAAG AAATAGCTGG GGTGAAGCGT GGGGAGAGAAAGGATACATC 1380 AAACTAGCTC TTCATTCTGG AAAGAAGGGA ACATGTGGTA TATTGGTTGAGCCAGTGTAT 1440 CCAGTGATTA ATCAATCAAT ATAAGCATTT CAGTGTTTGA CTAAGTAATTCTAATATATT 1500 TCAGCATTCT CAGAGATAAT TTTAGTTCAA ATGAACAATC TATTCATATATATAAGCATT 1560 CCATACTTAA TTATTTATTG ATTTTAATAA AATGTTTGGC TAAAGAAAGCAATCAAGATA 1620 ATTTATGGAC GTTCTATTGT TCTTACTTCA ATAATAATCC TTT 1663 534base pairs nucleic acid double linear DNA Cryptosporidium parvum 2TTAAGTAAAA TGGACATAGG AAACAACGTG GAAGAACATC AGGAATATAT TTCTGGACCA 60TACATTGCAT TAATTAATGG CACTAATCAA CAAAGGGAAC CGAATAAAAA GTTGAAAAAC 120ATAATAATTG CAACGTTGAT TGCAATCTTT ATAGTTTTGG TTGTTACTGT ATCTTTGTAT 180ATTACTAATA ACACCAGTGA CAAAATTGAC GATTTCGTAC CTGGTGATTA TGTTGATCCA 240GCAACTAGGG AGTATAGAAA GAGTTTTGAG GAGTTCAAAA AGAAATACCA CAAAGTATAT 300AGCTCTATGG AGGAGGAAAA TCAAAGATTT GAAATTTATA AGCAAAATAT GAACTTTATT 360AAAACAACAA ATAGCCAAGG ATTCAGTTAT GTGTTAGAAA TGAATGAATT TGGTGATTTG 420TCGAAAGAAG AGTTTATGGC AAGATTCACA GGATATATAA AAGATTCCAA AGATGATGAA 480AGGGTATTTA AGTCAAGTAG AGTCTCAGCA AGCGAATCAG AAGAGGAATT TGTT 534 678 basepairs nucleic acid single linear DNA Cryptosporidium parvum 3 CCCCCAAATTCTATTAATTG GGTGGAAGCT GGATGCGTGA ACCCAATAAG AAATCAAAAG 60 AATTGTGGGTCATGTTGGGC TTTCTCTGCT GTTGCAGCTT TGGAGGGAGC AACGTGTGCT 120 CAAACAAACCGAGGATTACC AAGCTTGAGT GAACAGCAAT TTGTTGATTG CAGTAAACAA 180 AATGGCAACTTTGGATGTGA TGGAGGAACA ATGGGATTGG CTTTTCAGTA TGCAATTAAG 240 AACAAATATTTATGTACTAA TGATGATTAC CCTTACTTTG CTGAGGAAAA AACATGTATG 300 GATTCATTTTGCGAGAATTA TATAGAGATT CCTGTAAAAG CCTACAAATA TGTATTTCCG 360 AGAAATATTAATGCATTAAA GACTGCTTTG GCTAAGTATG GACCAATTTC AGTTGCAATT 420 CAGGCCGATCAAACCCCTTT CCAGTTTTAT AAAAGTGGAG TATTCGATGC TCCTTGTGGA 480 ACCAAGGTTAATCATGGAGT TGTTCTAGTT GAATATGATA TGGATGAAGA TACTAATAAA 540 GAATATTGGCTAGTAAGAAA TAGCTGGGGT GAAGCGTGGG GAGAGAAAGG ATACATCAAA 600 CTAGCTCTTCATTCTGGAAA GAAGGGAACA TGTGGTATAT TGGTTGAGCC AGTGTATCCA 660 GTGATTAATCAATCAATA 678 401 amino acids amino acids single linear proteinCryptosporidium parvum 4 Met Asp Ile Gly Asn Asn Val Glu Glu His Gln GluTyr Ile Ser 1 5 10 15 Gly Pro Tyr Ile Ala Leu Ile Asn Gly Thr Asn GlnGln Arg Glu 20 25 30 Pro Asn Lys Lys Leu Lys Asn Ile Ile Ile Ala Thr LeuIle Ala 35 40 45 Ile Phe Ile Val Leu Val Val Thr Val Ser Leu Tyr Ile ThrAsn 50 55 60 Asn Thr Ser Asp Lys Ile Asp Asp Phe Val Pro Gly Asp Tyr Val65 70 75 Asp Pro Ala Thr Arg Glu Tyr Arg Lys Ser Phe Glu Glu Phe Lys 8085 90 Lys Lys Tyr His Lys Val Tyr Ser Ser Met Glu Glu Glu Asn Gln 95 100105 Arg Phe Glu Ile Tyr Lys Gln Asn Met Asn Phe Ile Lys Thr Thr 110 115120 Asn Ser Gln Gly Phe Ser Tyr Val Leu Glu Met Asn Glu Phe Gly 125 130135 Asp Leu Ser Lys Glu Glu Phe Met Ala Arg Phe Thr Gly Tyr Ile 140 145150 Lys Asp Ser Lys Asp Asp Glu Arg Val Phe Lys Ser Ser Arg Val 155 160165 Ser Ala Ser Glu Ser Glu Glu Glu Phe Val Pro Pro Asn Ser Ile 170 175180 Asn Trp Val Glu Ala Gly Cys Val Asn Pro Ile Arg Asn Gln Lys 185 190195 Asn Cys Gly Ser Cys Trp Ala Phe Ser Ala Val Ala Ala Leu Glu 200 205210 Gly Ala Thr Cys Ala Gln Thr Asn Arg Gly Leu Pro Ser Leu Ser 215 220225 Glu Gln Gln Phe Val Asp Cys Ser Lys Gln Asn Gly Asn Phe Gly 230 235240 Cys Asp Gly Gly Thr Met Gly Leu Ala Phe Gln Tyr Ala Ile Lys 245 250255 Asn Lys Tyr Leu Cys Thr Asn Asp Asp Tyr Pro Tyr Phe Ala Glu 260 265270 Glu Lys Thr Cys Met Asp Ser Phe Cys Glu Asn Tyr Ile Glu Ile 275 280285 Pro Val Lys Ala Tyr Lys Tyr Val Phe Pro Arg Asn Ile Asn Ala 290 295300 Leu Lys Thr Ala Leu Ala Lys Tyr Gly Pro Ile Ser Val Ala Ile 305 310315 Gln Ala Asp Gln Thr Pro Phe Gln Phe Tyr Lys Ser Gly Val Phe 320 325330 Asp Ala Pro Cys Gly Thr Lys Val Asn His Gly Val Val Leu Val 335 340345 Glu Tyr Asp Met Asp Glu Asp Thr Asn Lys Glu Tyr Trp Leu Val 350 355360 Arg Asn Ser trp Gly Glu Ala Trp Gly Glu Lys Gly Tyr Ile Lys 365 370375 Leu Ala Leu His Ser Gly Lys Lys Gly Thr Cys Gly Ile Leu Val 380 385390 Glu Pro Val Tyr Pro Val Ile Asn Gln Ser Ile 395 400 175 amino acidsamino acid single linear protein Cryptosporidium parvum 5 Met Asp IleGly Asn Asn Val Glu Glu His Gln Glu Tyr Ile Ser 1 5 10 15 Gly Pro TyrIle Ala Leu Ile Asn Gly Thr Asn Gln Gln Arg Glu 20 25 30 Pro Asn Lys LysLeu Lys Asn Ile Ile Ile Ala Thr Leu Ile Ala 35 40 45 Ile Phe Ile Val LeuVal Val Thr Val Ser Leu Tyr Ile Thr Asn 50 55 60 Asn Thr Ser Asp Lys IleAsp Asp Phe Val Pro Gly Asp Tyr Val 65 70 75 Asp Pro Ala Thr Arg Glu TyrArg Lys Ser Phe Glu Glu Phe Lys 80 85 90 Lys Lys Tyr His Lys Val Tyr SerSer Met Glu Glu Glu Asn Gln 95 100 105 Arg Phe Glu Ile Tyr Lys Gln AsnMet Asn Phe Ile Lys Thr Thr 110 115 120 Asn Ser Gln Gly Phe Ser Tyr ValLeu Glu Met Asn Glu Phe Gly 125 130 135 Asp Leu Ser Lys Glu Glu Phe MetAla Arg Phe Thr Gly Tyr Ile 140 145 150 Lys Asp Ser Lys Asp Asp Glu ArgVal Phe Lys Ser Ser Arg Val 155 160 165 Ser Ala Ser Glu Ser Glu Glu GluPhe Val 170 175 226 amino acids amino acid single linear proteinCryptosporidium parvum 6 Pro Pro Asn Ser Ile Asn Trp Val Glu Ala Gly CysVal Asn Pro 1 5 10 15 Ile Arg Asn Gln Lys Asn Cys Gly Ser Cys Trp AlaPhe Ser Ala 20 25 30 Val Ala Ala Leu Glu Gly Ala Thr Cys Ala Gln Thr AsnArg Gly 35 40 45 Leu Pro Ser Leu Ser Glu Gln gln Phe Val Asp Cys Ser LysGln 50 55 60 Asn Gly Asn Phe Gly Cys Asp Gly Gly Thr Met Gly Leu Ala Phe65 70 75 Gln Tyr Ala Ile Lys Asn Lys Tyr Leu Cys Thr Asn Asp Asp Tyr 8085 90 Pro Tyr Phe Ala Glu Glu Lys Thr Cys Met Asp Ser Phe Cys Glu 95 100105 Asn Tyr Ile Glu Ile Pro Val Lys Ala Tyr Lys Tyr Val Phe Pro 110 115120 Arg Asn Ile Asn Ala Leu Lys Thr Ala Leu Ala Lys Tyr Gly Pro 125 130135 Ile Ser Val Ala Ile Gln Ala Asp Gln Thr Pro Phe Gln Phe Tyr 140 145150 Lys Ser Gly Val Phe Asp Ala Pro Cys Gly Thr Lys Val Asn His 155 160165 Gly Val Val Leu Val Glu Tyr Asp Met Asp Glu Asp Thr Asn Lys 170 175180 Glu Tyr Trp Leu Val Arg Asn Ser Trp Gly Glu Ala Trp Gly Glu 185 190195 Lys Gly Tyr Ile Lys Leu Ala Leu His Ser Gly Lys Lys Gly Thr 200 205210 Cys Gly Ile Leu Val Glu Pro Val Tyr Pro Val Ile Asn Gln Ser 215 220225 Ile 226 345 amino acids amino acid single linear protein Carica 7Met Ala Met Ile Pro Ser Ile Ser Lys Leu Leu Phe Val Ala Ile 5 10 15 CysLeu Phe Val Tyr Met Gly Leu Ser Phe Gly Asp Phe Ser Ile 20 25 30 Val GlyTyr Ser Gln Asn Asp Leu Thr Ser Thr Glu Arg Leu Ile 35 40 45 Gln Leu PheGlu Ser Trp Met Leu Lys His Asn Lys Ile Tyr Lys 50 55 60 Asn Ile Asp GluLys Ile Tyr Arg Phe Glu Ile Phe Lys Asp Asn 65 70 75 Leu Lys Tyr Ile AspGlu Thr Asn Lys Lys Asn Asn Ser Tyr Trp 80 85 90 Leu Gly Leu Asn Val PheAla Asp Met Ser Asn Asp Glu Phe Lys 95 100 105 Glu Lys Tyr Thr Gly SerIle Ala Gly Asn Tyr Thr Thr Thr Glu 110 115 120 Leu Ser Tyr Glu Glu ValLeu Asn Asp Gly Asp Val Asn Ile Pro 125 130 135 Glu Tyr Val Asp Trp ArgGln Lys Gly Ala Val Thr Pro Val Lys 140 145 150 Asn Gln Gly Ser Cys GlySer Cys Trp Ala Phe Ser Ala Val Val 155 160 165 Thr Ile Glu Gly Ile IleLys Ile Arg Thr Gly Asn Leu Asn Glu 170 175 180 Tyr Ser Glu Gln Glu LeuLeu Asp Cys Asp Arg Arg Ser Tyr Gly 185 190 195 Cys Asn Gly Gly Tyr ProTrp Ser Ala Leu Gln Leu Val Ala Gln 200 205 210 Tyr Gly Ile His Tyr ArgAsn Thr Tyr Pro Tyr Glu Gly Val Gln 215 220 225 Arg Tyr Cys Arg Ser ArgGlu Lys Gly Pro Tyr Ala Ala Lys Thr 230 235 240 Asp Gly Val Arg Gln ValGln Pro Tyr Asn Glu Gly Ala Leu Leu 245 250 255 Tyr Ser Ile Ala Asn GlnPro Val Ser Val Val Leu Glu Ala Ala 260 265 270 Gly Lys Asp Phe Gln LeuTyr Arg Gly Gly Ile Phe Val Gly Pro 275 280 285 Cys Gly Asn Lys Val AspHis Ala Val Ala Ala Val Gly Tyr Gly 290 295 330 Pro Asn Tyr Ile Leu IleLys Asn Ser Trp Gly Thr Gly Trp Gly 305 310 315 Glu Asn Gly Tyr Ile ArgIle Lys Arg Gly Thr Gly Asn Ser Tyr 320 325 330 Gly Val Cys Gly Leu TyrThr Ser Ser Phe Tyr Pro Val Lys Asn 335 340 345 244 amino acids aminoacid single linear protein Plasmodium vinckei 8 Phe Pro Asp Ser Arg AspTyr Arg Ser Lys Phe Asn Phe Leu Pro 5 10 15 Pro Lys Asp Gln Gly Asn CysGly Ser Cys trp Ala Phe Ala Ala 20 25 30 Ile Gly Asn Phe Glu Tyr Leu TyrVal His Thr Arg His Glu Met 35 40 45 Pro Ile Ser Phe Ser Glu Gln Gln MetVal Asp Cys Ser Thr Glu 50 55 60 Asn Tyr Gly Cys Asp Gly Gly Asn Pro PheTyr Ala Phe Leu Tyr 65 70 75 Met Ile Asn Asn Gly Val Cys Leu Gly Asp GluTyr Pro Tyr Lys 80 85 90 Gly His Glu Asp Phe Phe Cys Leu Asn Tyr Arg CysSer Leu Leu 95 100 105 Gly Arg Val His Phe Ile Gly Asp Val Lys Pro AsnGlu Leu Ile 110 115 120 Met Ala Leu Asn Tyr Val Gly Pro Val Thr Ile AlaVal Gly Ala 125 130 135 Ser Glu Asp Phe Val Leu Tyr Ser Gly Gly Val PheAsp Gly Glu 140 145 150 Cys Asn Pro Glu Leu Asn His Ser Val Leu Leu ValGly Tyr Gly 155 160 165 Gln Val Lys Lys Ser Leu Ala Phe Glu Asp Ser HisSer Asn Val 170 175 180 Asp Ser Asn Leu Ile Lys Lys Tyr Lys Glu Asn IleLys Gly Asp 185 190 195 Asp Asp Asp Asp Ile Ile Tyr Tyr Trp Ile Val ArgAsn Ser Trp 200 205 210 Gly Pro Asn Trp Gly Glu Gly Gly Tyr Ile Arg IleLys Arg Asn 215 220 225 Lys Ala Gly Asp Asp Gly Phe Cys Gly Val Gly SerAsp Val Phe 230 235 240 Phe Pro Ile Tyr 244 29 base pairs nucleic acidsingle linear synthetic oligonucleotide Y is C/T W is A/T S is C/G 9AAAGGATCCT GYGGNWSNTG YTGGGCNTT 29 33 base pairs nucleic acid singlelinear synthetic oligonucleotide S is C/G K is G/T W is A/T R is A/G 10TTTGAATTCC CANSWRTTNY KNAYNATCCA RTA 33 24 base pairs nucleic acidsingle linear synthetic oligonucleotide 11 CCAGGTACCA TGGACATAGG AAAC 2424 base pairs nucleic acid single linear synthetic oligonucleotide YES12 CCCTCTAGAT GCTTATATTG ATTG 24 7 amino acids amino acid single linearpeptide 13 Cys Gly Ser Cys Trp Ala Phe 5 7 8 amino acids amino acidsingle linear peptides Xaa at 4 is Val/Ile Xaa at 5 is Lys/Arg 14 TyrTrp Ile Xaa Xaa Asn Ser Trp 5 8 5 amino acids amino acid single linearpeptide 15 Val Arg Asn Ser Trp 5 1203 base pairs nucleic acid doublelinear DNA Cryptosporidium parvum 16 ATGGACATAG GAAACAACGT GGAAGAACATCAGGAATATA TTTCTGGACC ATACATTGCA 60 TTAATTAATG GCACTAATCA ACAAAGGGAACCGAATAAAA AGTTGAAAAA CATAATAATT 120 GCAACGTTGA TTGCAATCTT TATAGTTTTGGTTGTTACTG TATCTTTGTA TATTACTAAT 180 AACACCAGTG ACAAAATTGA CGATTTCGTACCTGGTGATT ATGTTGATCC AGCAACTAGG 240 GAGTATAGAA AGAGTTTTGA GGAGTTCAAAAAGAAATACC ACAAAGTATA TAGCTCTATG 300 GAGGAGGAAA ATCAAAGATT TGAAATTTATAAGCAAAATA TGAACTTTAT TAAAACAACA 360 AATAGCCAAG GATTCAGTTA TGTGTTAGAAATGAATGAAT TTGGTGATTT GTCGAAAGAA 420 GAGTTTATGG CAAGATTCAC AGGATATATAAAAGATTCCA AAGATGATGA AAGGGTATTT 480 AAGTCAAGTA GAGTCTCAGC AAGCGAATCAGAAGAGGAAT TTGTTCCCCC AAATTCTATT 540 AATTGGGTGG AAGCTGGATG CGTGAACCCAATAAGAAATC AAAAGAATTG TGGGTCATGT 600 TGGGCTTTCT CTGCTGTTGC AGCTTTGGAGGGAGCAACGT GTGCTCAAAC AAACCGAGGA 660 TTACCAAGCT TGAGTGAACA GCAATTTGTTGATTGCAGTA AACAAAATGG CAACTTTGGA 720 TGTGATGGAG GAACAATGGG ATTGGCTTTTCAGTATGCAA TTAAGAACAA ATATTTATGT 780 ACTAATGATG ATTACCCTTA CTTTGCTGAGGAAAAAACAT GTATGGATTC ATTTTGCGAG 840 AATTATATAG AGATTCCTGT AAAAGCCTACAAATATGTAT TTCCGAGAAA TATTAATGCA 900 TTAAAGACTG CTTTGGCTAA GTATGGACCAATTTCAGTTG CAATTCAGGC CGATCAAACC 960 CCTTTCCAGT TTTATAAAAG TGGAGTATTCGATGCTCCTT GTGGAACCAA GGTTAATCAT 1020 GGAGTTGTTC TAGTTGAATA TGATATGGATGAAGATACTA ATAAAGAATA TTGGCTAGTA 1080 AGAAATAGCT GGGGTGAAGC GTGGGGAGAGAAAGGATACA TCAAACTAGC TCTTCATTCT 1140 GGAAAGAAGG GAACATGTGG TATATTGGTTGAGCCAGTGT ATCCAGTGAT TAATCAATCA 1200 ATA 1203

1-18. (Cancelled)
 19. An antibody binding to a Cryptosporidiumcryptopain antigen comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 4 and SEQ ID NO:
 6. 20. The antibody ofclaim 19, binding to the amino acid sequence of SEQ ID NO:
 6. 21. Theantibody of claim 19, wherein the antibody is selected from the groupconsisting of monoclonal and polyclonal.
 22. The antibody of claim 19detecting a presence of Crytosporidium by formation of anantibody-antigen complex.
 23. The antibody of claim 22 wherein theantibody is polyclonal.
 24. The antibody of claim 22 wherein theantibody is monoclonal.
 25. A method of treatment of Crytosporidiuminfections comprising administering to a subject in need of suchtreatment either an anti-Crytosporidium antibody binding to a proteincomprising a sequence of SEQ ID NO: 4, SEQ ID NO: 6 or a fragmentthereof, or a DNA or RNA vaccine comprising a DNA sequence identified asSEQ ID NO: 1 or a fragment thereof.
 26. The method of claim 25 whereinthe antibody is polyclonal.
 27. The method of claim 25 wherein theantibody is monoclonal.
 28. The method of claim 25 comprisingadministration of an anti-Crytosporidium vaccine comprising theCrytosporidium antigen cryptopain comprising the amino acid sequence ofeither SEQ ID NO: 5 or SEQ ID NO:
 6. 29. The method of claim 25 whereinthe vaccine further comprises an adjuvant.
 30. The method of claim 28wherein the vaccine comprises the amino acid sequence of SEQ ID NO: 5.31. The method of claim 28 wherein the vaccine comprises the amino acidsequence of SEQ ID NO:
 6. 32. The method of claim 25 wherein the vaccinecomprises the DNA sequence of SEQ ID NO: 1 or the fragment thereofidentified as SEQ ID NO: 2 or SEQ ID NO:
 3. 33. The method of claim 25wherein the vaccine comprises the DNA sequence identified as SEQ IDNO:
 1. 34. The method of claim 25 wherein the vaccine comprises the DNAfragment identified as SEQ ID NO:
 2. 35. The method of claim 25 whereinthe vaccine comprises the DNA fragment identified as SEQ ID NO:
 3. 36. Amethod of diagnosing Crytosporidium infection, comprising: (a)contacting a sample of a body specimen, fluid or tissue obtained from asubject, with an anti-Crytosporidium antibody having specificity for anantigen identified by an amino acid sequence of either SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, or a fragment thereof; and (b) detecting aformation of an antibody/antigen complex in the sample.
 37. The methodof claim 36 wherein the antibody binds to the antigen identified by theamino acid sequence of SEQ ID NO:
 4. 38. The method of claim 36 whereinthe antibody binds to the antigen identified by the amino acid sequenceof SEQ ID NO:
 5. 39. The method of claim 36 wherein the antibody bindsto the antigen identified by the amino acid sequence of SEQ ID NO: 6.40. A polyclonal anti-cryptopain antibody suitable for treatment ofCrytosporidium infections wherein said antibody is administered to aperson in need of such treatment, said antibody produced against arecombinant Crytosporidium cryptopain antigen having an amino acidsequence of either SEQ ID NO: 4 or SEQ ID NO: 6, said antibody bindingto said cryptopain antigen, wherein said antibody is administered in anamount effective to provide immunity or to inhibit existingCrytosporidium infection.
 41. The antibody of claim 40 raised against acryptopain antigen having an amino acid sequence of SEQ ID NO:
 4. 42.The antibody of claim 41 raised against a cryptopain antigen having anamino acid sequence of SEQ ID NO:
 6. 43. A polyclonal or monoclonalanti-Crytosporidium antibody suitable for detecting Crytosporidium in abiological or environmental sample wherein said antibody is contactedwith a biological sample specimen or with an environmental samplesuspected to be infected with Crytosporidium, wherein said antibody isproduced against a recombinant Crytosporidium cryptopain antigen havingamino acid sequence of either SEQ ID NO: 4 or SEQ ID NO: 6, and binds tocryptopain antigen of either SEQ ID NO: 4 or SEQ ID NO:
 6. 44. Theantibody of claim 43 in an anti-cryptopain antibody/cryptopain antigencomplex in the biological or environmental sample.
 45. The antibody ofclaim 43 wherein the anti-cryptopain antibody/cryptopain antigen complexis detected with either an enzyme-linked immunoassay or with aradioactive assay.
 46. The antibody of claim 45 wherein the formation ofthe anti-cryptopain antibody/cryptopain antigen complex is detected bysolid phase method, with double antibody assay, sandwich double antibodyassay or triple antibody assay or ELISA.
 47. The antibody of claim 45which detects a presence of the anti-cryptopain antibody/cryptopainantigen complex of Crytosporidium in a tissue, fluid, or biologicalsample.
 48. The antibody of claim 43 which detects a presence of theanti-cryptopain antibody/cryptopain antigen complex in a stool, urine,saliva, blood or serum sample specimen.
 49. The antibody of claim 43wherein the antibody binds specifically to SEQ ID NO: 6.